Elucidating the distinct contributions of miR-122 in the HCV life cycle reveals insights into virion assembly

Abstract Efficient hepatitis C virus (HCV) RNA accumulation is dependent upon interactions with the human liver-specific microRNA, miR-122. MiR-122 has at least three roles in the HCV life cycle: it acts as an RNA chaperone, or ‘riboswitch’, allowing formation of the viral internal ribosomal entry site; it provides genome stability; and promotes viral translation. However, the relative contribution of each role in HCV RNA accumulation remains unclear. Herein, we used point mutations, mutant miRNAs, and HCV luciferase reporter RNAs to isolate each of the roles and evaluate their contribution to the overall impact of miR-122 in the HCV life cycle. Our results suggest that the riboswitch has a minimal contribution in isolation, while genome stability and translational promotion have similar contributions in the establishment phase of infection. However, in the maintenance phase, translational promotion becomes the dominant role. Additionally, we found that an alternative conformation of the 5′ untranslated region, termed SLIIalt, is important for efficient virion assembly. Taken together, we have clarified the overall importance of each of the established roles of miR-122 in the HCV life cycle and provided insight into the regulation of the balance between viral RNAs in the translating/replicating pool and those engaged in virion assembly.

the QuikChange XL II kit (Agilent) according to manufacturer's instructions. All plasmid sequences were verified by Sanger sequencing (Génome Québec).

In Vitro Transcription
To make A-capped 6X BoxB and 6X scrambled (scr) mRNAs, the plasmids were linearized with BamHI and in vitro transcribed using T7 RNA polymerase (NEB). Briefly, 1 µg of template GlycoBlue TM co-precipitant (ThermoFisher Scientific) and stored at -80°C until use.

Tethering Assays
For hAgo2 tethering assays, 1 × 10 6 HEK 293T cells were seeded in each well of three 6-well BoxB or 6X scr mRNAs and RLuc control mRNA using 2 µL DMRIE-C reagent (ThermoFisher Scientific) in 400 µL Opti-MEM per well. Transfection complexes were left on the cells overnight, and cells were harvested in 100 µL 1X PLB 16 h post-transfection. Experiments were conducted in three independent replicates with two technical replicates (two wells) per condition within each experiment.

Western Blot Analysis
Whole cell lysates were prepared in 1X passive lysis buffer (Promega) and stored at -80 °C until use. Lysates were cleared by centrifugation at 16,000 × g and supernatant protein concentration was assessed by Bradford assay using the Pierce Coomassie assay kit (ThermoFisher Scientific) according to manufacturer's instructions, with the modification that 1-2 µL of supernatant and 5 µL of standard was used with 200 µL of Coomassie protein assay reagent. Ten micrograms of protein was loaded on 10% SDS-PAGE gels and run at 80 V for 20 min, followed by 100V.

In Vitro Selective 2´ Hydroxyl Acylation Analyzed by Primer Extension (SHAPE)
In vitro SHAPE analysis was performed in quadruplicate as previously described (2). Briefly, 5 pmol of HCV 5´ UTR RNA was re-folded and incubated in SHAPE buffer (333 mM HEPES, pH 8.0; 20 mM MgCl2; 333 mM NaCl) for 1 h at 37ºC. RNA was then exposed to 0.01 M NAI-N3 or dimethyl sulfoxide (DMSO) (treatment control) for 5 min at 37ºC, and then extracted using TRIzol reagent (ThermoFisher Scientific) according to the manufacturer's instructions. Extracted labelled RNA was precipitated and stored at -80ºC. Labelled RNA was used for SHAPE analysis by capillary electrophoresis as previously described (3).

5´ Rapid Amplification of cDNA Ends (RACE)
Reverse transcription. Huh-7.5 cells from each well of a 6-well plate were lysed in 500 µL TRIzol reagent (ThermoFisher Scientific). Samples were stored at -80°C until use and RNA was extracted using chloroform, according to the manufacturer's instructions. Reverse transcription of the 5´ end of the genome was performed using the HCV-specific primer HCV_RACE_RT (5´ -CGC GCG GTC CGC CGG GTA GAA TTC/iSp18/GCC CGG AAA CTT AAC GTC TT -3´) which had previously been 5´ phosphorylated with T4 polynucleotide kinase (T4 PNK, NEB) according to manufacturer's instructions, with the modification that the 37°C incubation was increased to 1 h.
Briefly, 1 µg total RNA was incubated with 100 nM monophosphorylated primer and dNTP mix (1 mM each) at 95°C for 5 min to remove any secondary structure. The reaction mixture was then placed on ice and First Strand buffer, DTT to 10 µM, 20 U RiboLock RNase inhibitor and 200 U SuperScript III reverse transcriptase were added. The mixture was then incubated at 53°C for 30 min to allow for cDNA synthesis, and the enzyme was heat inactivated at 70°C for 15 min. Eight microliters of a 0.5 N NaOH/0.25 M EDTA solution was added, and the reaction mixture was heated to 65°C for 15 min to hydrolyze the template RNA. Water was added to 200 µL total and the cDNA was then purified by ethanol precipitation.
cDNA circularization. After ethanol precipitation, the cDNA was resuspended in 7.3 µL water.
One microliter of DMSO was added and the mixture was heated at 95°C for 5 min to denature secondary structures, followed by a 1 min incubation on ice and quick centrifugation. The following components were added as a master mix: T4 RNA ligase buffer, 1 µM ATP, 22.5% PEG 8000, 5 U T4 RNA ligase 1 (high concentration, NEB). The reaction mixture was mixed by gentle pipetting, and an additional 10 U of T4 RNA ligase 1 was added, for a total volume of 20 µL. The reaction was incubated at room temperature overnight, with gentle pipetting every 30 min for approximately 2-3 h at the onset of the reaction. Sequencing. The Gibson assembly ligation reaction was transformed into NEB stable or 10 beta cells and at least 10 colonies were screened per experimental replicate (33 colonies total). Plasmid DNA was purified using the QIAprep spin miniprep kit (Qiagen) according to manufacturer's instructions, and Sanger sequencing was performed at Génome Québec. Figure S1. Independent biological replicate (GNN) data for the overall contributions of miR-122's riboswitch, genome stability, and translation promotion activities. (A-D) Full-length RLuc HCV (GNN) RNAs were co-electroporated into miR-122 knockout (KO) cells with miR-122 or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activity was monitored over time. In (C) and (D), the 48-72 h time points were lost due to an error during sample preparation. All four independent biological replicates (A-D) were used to calculate the fold-change in Figure 1C. The limit of detection is indicated. RLuc activity was monitored over time. All six independent biological replicates (A-F) were used to calculate the fold-change in Figure 1F. The limit of detection is indicated. RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activities for (A-B) S1+S2:p3 (GNN) and (D-E) S1+S2:p3 viral RNAs were monitored over time. The limit of detection is indicated. Data in (A) and (D) is representative data from one of three independent biological replicates with three technical replicates, and error bars represent the standard error of the mean (SEM). The limit of detection is indicated. In (B) and (E), RLuc activity for S1+S2:p3 (GNN) or S1+S2:p3 HCV RNAs at 6 h or 72 h, respectively, normalized to the FLuc (transfection efficiency) control at 2 h, were used to calculate the fold change, with the control miRNA condition set to 1. Data is displayed as the mean of four (S1+S2:p3 GNN) and three (S1+S2:p3) independent biological replicates, and error bars represent the SEM. Viral RNA levels for (C) S1+S2:p3 (GNN) and (F) S1+S2:p3 HCV were monitored by RT-qPCR (as described in Figure 1). The limit of detection is indicated. Data is displayed as the mean of three independent biological replicates (except for the 6h S1+S2:p3 (GNN) + ctrl miRNA and 72h S1+S2:p3 (GNN) + miR-122 p3U conditions which represent two independent replicates), with error bars corresponding to the SEM. Statistical significance was determined by multiple Student's t test, ****p ≤ 0.0001; *** p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05 ns, not significant (p ≥ 0.05). Figure S4. Independent biological replicate (S1+S2:p3 GNN) data for the alternative quantification of the overall contribution of miR-122's riboswitch, genome stability, and translation promotion activities in Huh-7.5 cells. (A-D) Full-length RLuc HCV S1+S2:p3 (GNN) RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activity was monitored over time. In (B), the 24 h time point was lost due to an error during sample preparation. All four independent biological replicates (A-D) were used to calculate the foldchange in Figure S3B. The limit of detection is indicated. Figure S5. Independent biological replicate (S1+S2:p3) data for the alternative quantification of the overall contribution of miR-122's riboswitch, genome stability, and translation promotion activities in Huh-7.5 cells. (A-D) Full-length RLuc HCV S1+S2:p3 RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. Half the number of cells were plated for the 24-72 h time points and the 48-72 h lysates were diluted 2-fold to ensure values were within range of the luciferase assay. RLuc activity was monitored over time. In (D), the 72 h time point was lost due to an error during sample preparation. As such, three independent biological replicates (A-C) were used to calculate the fold-change in Figure S3E. The limit of detection is indicated.    in (B) is representative data from one of four independent biological replicates with three technical replicates, and error bars represent the standard error of the mean (SEM). The limit of detection is indicated. (C) RLuc activity at 6 h normalized to Fluc (transfection efficiency) control at 2 h, was used to calculate the fold change between WT (GNN) and G20A (GNN), with the WT (GNN) condition set to 1. Data is displayed as the mean of four independent biological replicates, and error bars represent the SEM. (D) Viral RNA levels were monitored by RT-qPCR as described in Figure 1. The limit of detection is indicated. Data is displayed as the mean of three independent biological replicates, with error bars corresponding to the SEM. Statistical significance was determined by multiple Student's t test. ns, not significant (p ≥ 0.05). All four independent biological replicates (A-D) were used to calculate the fold-change in Figure S9C. The limit of detection is indicated. Figure S11. Independent biological replicate (S1:p3 GNN) data for genome stability experiments. (A-E) Full-length RLuc S1:p3 (GNN) HCV RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activity was monitored over time. All five independent biological replicates (A-E) were used to calculate the fold-change in Figure 3C. The limit of detection is indicated. Figure S12. Independent biological replicate (S1:p3) data for genome stability experiments. (A-E) Full-length RLuc S1:p3 HCV RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activity was monitored over time. All five independent biological replicates (A-E) were used to calculate the fold-change in Figure  3F. The limit of detection is indicated.     Figure 1). The limit of detection is indicated. Data in (B) and (E), are representative data from one of four independent biological replicates with three technical replicates, and error bars represent the standard error of the mean (SEM). In (C) and (F), the RLuc activity for G20A S2:p3 (GNN) or G20A S2:p3 HCV RNAs at 6 h or 72 h, respectively, normalized to FLuc (transfection efficiency) control at 2 h, were used to calculate the fold change, with the control miRNA condition set to 1. The limit of detection is indicated. Data displayed is the mean of four independent biological replicates, and error bars represent the SEM. Viral RNA levels for (D) G20A S2:p3 (GNN) and (G) G20A S2:p3 HCV were monitored by RT-qPCR (as in described in Figure 1). The limit of detection is indicated. Data is displayed as the mean of three independent biological replicates with error bars corresponding to the SEM. Statistical significance was determined by multiple Student's t test, ****p ≤ 0.0001; *** p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05 ns, not significant (p ≥ 0.05). Figure S17. Independent biological replicate (G20A S2:p3 GNN) data for translational promotion experiments. (A-D) Full-length RLuc G20A S2:p3 GNN HCV RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activity was monitored over time. All four independent biological replicates (A-D) were used to calculate the fold-change in Figure S16C. The limit of detection is indicated. Full-length RLuc G20A S2:p3 HCV RNAs were co-electroporated into Huh-7.5 cells with miR-122 p3U or control (ctrl) miRNA, and a capped Firefly luciferase (FLuc) reporter RNA. RLuc activity was monitored over time. All four independent biological replicates (A-D) were used to calculate the fold-change in Figure S16F. The limit of detection is indicated.